In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways.

Yusuke Karasawa,Miki Nonaka,Takaaki Mizuguchi,Yui Kuroda,Masako Iseki,Kanako Miyano,Yasuhito Uezono,Hideaki Fujii,Masakazu Hayashida,Akane Komatsu,Kaori Ohshima,Masahiro Yamaguchi,Keisuke Yamaguchi

doi:10.3390/molecules26196079

Abstract

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

Highlights

Opioid analgesics are widely used as key medications for relief from pain, including perioperative pain, cancer pain, and nonmalignant chronic pain
Opioid receptors (ORs), which belong to the G-protein-coupled receptor (GPCR) family [3], are classified into three subtypes—μ (MOR), δ (DOR), and κ (KOR)—and opioid analgesics mainly bind to MOR to exert their effects [4]
Internal signals from ORs are transmitted through two major pathways after the ligand conjugates with Gi/o proteins, followed by internalization of membrane receptors; the two pathways are the G-protein-mediated pathway that is required for analgesia, which is induced by decreasing the intracellular cAMP levels, and the β-arrestin-mediated pathway, which is associated with side effects [5,6]

Summary

Introduction

Opioid analgesics are widely used as key medications for relief from pain, including perioperative pain, cancer pain, and nonmalignant chronic pain. A biased analgesic with a pharmacological profile of favoring the activation of the G protein-mediated pathway over that of the β-arrestin-mediated pathway is desirable because it is considered to be effective and has fewer adverse events [7,8]. From this perspective, some molecules have been studied and indicated as G-protein-biased agonists in the past decades [9,10]. TRV130 (oliceridine) has been evaluated by intravenous administration in clinical studies and was approved as the first G-protein-biased agonist that can be used in clinical practice [11]

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Results

Conclusion